In modern surgery, scaffolds are increasingly used, which are implantable devices that have the function of temporarily compensating the impaired functionality of body parts and tissues, and which are then colonized by the natural cellular regrowth of the damaged part or tissue to achieve the regeneration of the same.
The materials for producing the scaffolds can be very different, depending on the intended application. For example, inorganic scaffolds consisting of mixtures of hydroxyapatite and β-tricalcium phosphate are typically used for the temporary replacement and regrowth of bone tissue. However, polymeric scaffolds are far more common, preferably based on biodegradable and biocompatible biopolymers or synthetic polymers, intended to temporarily replace non-rigid tissues.
A polymeric scaffold must possess a series of surface and bulk properties optimized for the function to be carried out in vivo. Among the properties of interest we may consider the morphological characteristics at a nano, micro and macroscopic level; the physical-mechanical properties and performance (ideally, these should be as close as possible to the in vivo characteristics and performance of the tissues to be regenerated); and the chemical and biological properties, with particular reference to biocompatibility (i.e. the ability of supporting adhesion and cell growth, not causing inflammatory and/or immunogenic reactions, not releasing hazardous substances, etc.) and to biodegradability or bioresorbability (which must be commensurate with the residence time of the device in vivo, which in turn depends on the reconstruction rate of the tissue to be repaired). Other properties of interest can be porosity, permeability to fluids, ability to uptake, retain and then release, when required, active agents, growth factors or drugs, etc. to the implantation site.
A particularly promising natural polymer for use in the production of scaffolds is fibroin, a silk protein produced in nature by Lepidoptera (domestic species: Bombyx mori; wild species: Antheraea pernyi, Philosamia ricini, etc.), other insects and arachnids. Fibroin can also be produced by recombinant DNA techniques. Fibroin is obtained from natural silk with the so-called “scouring” treatment, which consists in the removal of the sericin layer covering the fibroin; this treatment is generally carried out through a water bath optionally added with alkalis (soap), acids or enzymes, at temperatures between about 60 and 120° C., if necessary by operating in an autoclave. The fibroin thus obtained is in the form of microfibers having an average diameter of 12-14 μm, with an ultimate strength of about 600 MPa and elongation at break values of 25-30% (values referred to the microfiber of B. mori). Two- or three-dimensional structures (threads, yarns, woven fabrics, knitted fabrics, nonwoven fabrics, nets, braids, cords, etc.) can be produced with these microfibers, making use of technologies developed for silk applications in the textile field and which thus fall within the ordinary knowledge of the man skilled in the art.
The interest towards fibroin is mainly due to its proven biocompatibility, which is expressed through the ability of supporting the growth and proliferation of various cell types, the lack of immunogenic and inflammatory reactions, and the marked angiogenic properties, particularly useful in the case of the repair/regeneration of living tissues. In addition, the physical-mechanical characteristics mentioned above allow producing scaffolds with mechanical properties suitable for the purpose (in particular, high resistance to tensile, bending, compression stress; good elasticity; resilience, etc.); finally, the scaffolds made of fibroin have biodegradability characteristics in the medium-long term in vivo (from a few months to 1-2 years, depending on the characteristics of the biological environment of the implantation site), optimal for applications in which the scaffold must ensure mechanical support for prolonged times.
Thanks to these properties, fibroin has already been proposed in the art for the production of scaffolds.
The coupling of polymers in the form of fibers through partial dissolution and subsequent re-deposit of polymer on the fibers is known for example from patent application DE 1436311 A1.
U.S. Pat. No. 8,202,379 B1 describes the coupling of fibers of natural or synthetic polymers by treatment of the same with mixtures containing an ionic liquid and a second liquid compound, generally water, an alcohol or a ketone.
These documents only describe the coupling of fibers homogeneous in size and chemical, physical and mechanical properties (as derived from the same production and/or working process).
Patent applications WO 03/043486 A2, EP 2210971 A1 and WO 2011/031854 A1 describe fibroin structures intended for the reconstruction of ligaments (in particular the anterior cruciate ligament of the knee), consisting of a hierarchy of fibrous structures assembled at increasing levels up to reaching the dimensions and the mechanical performance required for the application.
Patent applications WO 2013/012635 A2, WO 2013/082093 A1 and WO 2012/111309 A1 describe instead devices based on native fibroin microfibers, having a net structure obtained by knitting, optimized for the reparative surgery of damaged tissues in the abdominal and pelvic areas, for plastic surgery of the breast, and for the realization of vascular prostheses, respectively.
These scaffolds are produced from fibroin microfibers (which as said have diameters of about 12-14 μm) through the use of textile technologies, in which the basic silk thread, consisting of at least 20 microfibers of fibroin, is generally assembled by doubling and twisting operations in hierarchically superior structures (“yarns”) whose transverse dimensions may range from a tenth of a millimeter up to one millimeter or more. Although the textile structures thus produced usually have excellent characteristics of softness and smoothness and at a macroscopic level they adapt easily to the surface to which they adhere, at a microscopic level they can display stiffness areas such as to cause local irritation/inflammation reactions; furthermore, due to their high crystallinity and toughness, fibroin microfibers are able to exert friction forces of such magnitude as to abrade the surface of the tissue with which they come into contact; these problems can lead, in the worst cases, to the partial or total failure of the implant. Another drawback of fibroin microfiber scaffolds is that they display unfavorable surface/volume ratios, so that a total area suitable for autologous colonization involves a relatively high load of material to be placed in the implantation site, with the consequences of a potential overload of physiological and metabolic activity due to a local accumulation of degradation products to be disposed of by the organism. Finally, the natural degradation times of the fibroin microfibers in some cases may be too long compared to the rate of neo-tissue formation, and thus interfere with its growth.
In order to obviate these drawbacks, it has been proposed the use of fibroin in the form of nanofibers, that is, having a diameter less than one micron and typically from a few tens up to a few hundred nanometers.
These nanofibers can be produced by known processes, in which the native fibroin is first solubilized in a suitable solvent, and then regenerated with processes such as force-spinning or electrospinning. In these processes, the solution of fibroin is passed through a capillary tube, called a spinneret, giving rise to a liquid filament of nanoscopic dimensions, which is accelerated towards a collector; in the case of force-spinning, the acceleration is caused by the centrifugal force (due to the rotation of the spinneret at a speed of several thousand rpm), while in the case of electrospinning, it is caused by a potential difference between the nozzle of the spinneret and a manifold, which loads the liquid thus causing the production of a jet of solution; thanks to the viscoelastic properties of the polymer, the jet undergoes a drawing process which, accompanied by the simultaneous evaporation of the solvent, leads to the production of the nanofiber which accumulates on the collector.
Recent studies have demonstrated the excellent properties of the scaffolds made with fibroin nanofibers.
The article “In vivo regeneration of elastic lamina on fibroin biodegradable vascular scaffold”, I. Cattaneo et al., Int. J. Artif. Organs 36 (2013) 166, shows that a tubular scaffold of electrospun fibroin, implanted in the abdominal portion of the aorta of the rat, allows the formation of a vascular tissue totally similar to the native one from the morphological and functional point of view. The article “Decellularized silk fibroin scaffold primed with adipose mesenchymal stromal cells improves wound healing in diabetic mice”, S. E. Navone et al., Stem Cell Research & Therapy, 5 (2014) 7, shows the effectiveness of electrospun fibroin patches, pre-activated by contact with mesenchymal cells of the adipose tissue, in inducing wound healing in diabetic mice through biological mechanisms involving the direct stimulation of angiogenic processes by the material.
Scaffolds have also been described made by coupling of fibroin microfibers and nanofibers.
Patent application CN 101879330 A describes a device, proposed as a vascular prosthesis and/or a guide for the regeneration of nerves, having a three-layers tubular structure, wherein the inner layer is a porous deposit made from regenerated fibroin, the intermediate layer is a tubular structure of woven microfibers of fibroin in the form of a net, and the outer layer consists of a nanofibrous fibroin structure produced by electrospinning. The production process of the device described in this document is complex. The inner layer is initially produced in tubular shape with standard weaving methods, the layer thus obtained is immersed in a solution of fibroin and the resulting intermediate product is dried at 40-60° C. This first intermediate product is fitted onto a collector pin for electrospinning, and a layer of nano-microfibrous fibroin is deposited on the outer surface of said first intermediate product mentioned above by means of this technique; the composite thus obtained is then immersed in methanol or ethanol for 1-4 hours. Finally, this first composite is introduced into a mold and the porous layer is produced on its inner tubular surface by deposit from a fibroin solution; the porosity of the innermost layer is obtained by a treatment at temperatures between −80° C. and −10° C. The three layers of this composite are bonded together by means of fibroin films that are formed on the surface of the same during the immersions in solvents or in fibroin solutions. The fibroin films produced according to the process of this document, however, once dried and crystallized are extremely fragile, such as to fracture immediately as they are urged by tensile, bending, compression, etc., mechanical stress; this can lead to the creation of morphological and mechanical discontinuity between the different layers which can easily lead to a loss of the geometric and performance characteristics, such as the yielding and/or the collapse of the weaker layers from the mechanical point of view (in particular the nanofibrous ones).
Patent application CN 102499800 A describes a device which may find application as a stent or prosthesis for the repair of small blood vessels, also in this case consisting of a hybrid structure with three layers; the inner and the intermediate layer are made of nanofibrous structures obtained by electrospinning of a fibroin/polycaprolactone mixture, while the outer layer consists of a tubular structure of fibroin microfibers, produced with a braiding machine. The three layers, produced separately and fitted one on top of the other as sleeves, are held together by a series of annular stitches. This device partly has the same drawbacks as the former one; furthermore, the fact that the coupling is realized with stitches spaced apart from one another leaves a partial freedom of movement to the various components; in stressing working conditions from the mechanical and biological point of view, such as those that can occur in the progress of the implantation in vivo, this could create local stresses of such a magnitude as to interfere with the regenerative processes in progress, especially if the material is exposed to flows of physiological fluids.
The object of the present invention is to provide a hybrid composite material made of fibroin micro- and nanofibers which allows producing scaffolds for medical applications free from the drawbacks of the prior art.